Citation
Jordens, I. (2005, November 23). Transport of Lysosome-Related Organelles. Retrieved from https://hdl.handle.net/1887/4341
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in theInstitutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/4341
Opposing motor activities of dynein and kinesin
determine retention and transport of MHC cl
ass
II-
containing compartments
Opposing motor activities of dynein and kinesin determine
retention and transport of M HC cl
ass II-containing
compartments
Richard Wubbolts, Mar Fernandez-Borja, Ingrid Jordens, Eric Reits, Simone Dusseljee, Christophe Echeverri, Richard B. Vallee and Jacques Neefjes
Keywords: Antigen presentation, Dynamitin, Dynein, M HC class II, Kinesin, Vesicular transport, M IIC
Summary
M HC class II molecules exert their function at the cell surface by presenting to T cells antigenic fragments thatare generated in the endosomalpathway.The class II molecules are targetted to early lysosomal structures, termed M IIC, where they interact with antigenic fragments and are subsequently transported to the cell surface. W e previously visualised vesicular transport of M HC class II-containing early lysosomes from the microtubule organising centre (M TOC) region towards the cellsurface in living cells.Here we show that the M IIC move bidirectionally in a ‘stop-and-go’ fashion. Overexpression of a motor head-deleted kinesin inhibited M IIC motility,showing thatkinesin is the motor thatdrives its plus end transport towards the cell periphery. Cytoplasmic dynein mediates the return of vesicles to the M TOC area and effectively retains the vesicles at this location, as assessed by inactivation of dynein by overexpression of dynamitin. Our data suggest a retention mechanism that determines the perinuclear accumulation of M IIC, which is the result of dynein activity being superior over kinesin activity. The bidirectional nature of M IIC movementis the resultof both kinesin and dynein acting reciprocally on the M IIC during its transport. The motors may be the ultimate targets of regulatory kinases since the protein kinase inhibitor staurosporine induces a massive release of lysosomal vesicles from the M TOC region thatis morphologically similar to thatobserved after inactivation of the dynein motor.
Introduction
M HC (M ajor Histocampatbility Complex) class II molecules associate with antigenic fragments in endocytic compartments where antigen degradation occurs (W atts, 1997). In most cell types antigen loading occurs in late endocytic, early lysosomal structures, called M IIC (M HC classII-containing compartment),to which class II molecules are targeted by the associated invariantchain (Ii;Cresswell,1994;W olf etal.,1996).Iiis degraded in a stepwise manner upon arrival in these structures (Blum et al., 1988; Nguyen and Humphreys, 1989; Pieters et al., 1991) but an Ii fragment, termed CLIP (carboxyterminal Ii leupeptin-induced peptide),escapes fulldegradation by occupying the class II peptide binding groove (Avva et al., 1994; Ghosh et al., 1995). This fragment is exchanged for other peptides by a specific chaperone called HLA-DM (Jensen,1998) which is also found in M IIC (Karlsson etal.,1994; Sanderson etal.,1994;Fernandez-Borja etal.,1996;Pierre etal.,1996;Robbins etal.,1996).
towards the cell surface using cells expressing class II tagged with the Green Fluorescent Protein (GFP; Heim and Tsien, 1996). The GFP-tagged class II molecules functioned identically to endogenous class II in assembly, peptide loading, intracellular transport and distribution (Wubbolts et al., 1996). Fluorescent class II molecules accumulate in structures localised in a perinuclear area around the Golgi and the microtubule organising centre (MTOC). The structures contain markers for class II-containing early lysosomal vesicles (MIIC; Peters et al., 1991), like HLA-DM, CD63, mature cathepsin D and acidic peptidases, while they lack the late endosomal marker mannose 6-phosphate receptor (Fernandez-Borja et al., 1996; Wubbolts et al., 1996; van Ham et al., 1997). Occasionally, a MIIC is released from the MTOC region and moves towards the plasma membrane. How the MIIC are retained in the perinuclear area and which factors determine their release are currently unknown and are studied here.
The regulation of class II transport may have many characteristics of the transport of specialised vesicular structures such as cytolytic granules, melanosomes or other storage vesicles (Griffiths and Argon, 1995; Gruenberg and Kreis, 1995). However, vesicular transport of class II in most cells, including Mel JuSo cells, does not seem to be tightly regulated, yet appears to be constitutive. In some cell types, examples of regulation in class II transport have been reported. Inhibition of Ii degradation inhibits class II loading and delays surface expression of newly synthesised class II molecules (Neefjes and Ploegh, 1992; Zachgo et al., 1992) and alters intracellular distribution of class II in dendritic cells (Pierre and Mellman, 1998). Differentiation of dendritic cells affects intracellular distribution of class II (Sallusto and Lanzavecchia, 1994; Cella et al., 1997) and its transport through endosomes (Pierre et al., 1997). Moreover, treatment of monocytes with IL-10 inhibits arrival of newly synthesised class II to the plasma membrane and maintains endocytosed class II intracellularly (Koppelman et al., 1997). In all cases the molecular effector mechanisms underlying these effects are unknown.
Many intracellular vesicles are transported along polarised cytoskeletal elements, microtubules, by utilising molecular motor proteins (reviewed in Vallee and Scheetz, 1996; Hirokawa, 1998). Kinesin moves towards the fast growing end (or plus end), away from the MTOC, and cytoplasmic dynein moves towards the minus end. The amino-terminal globular domains of the motors are involved in the binding to microtubules (Vale and Fletterick, 1997; Woehlke et al., 1997), with dynein having a finger-like protrusion from the large globule that interacts with the microtubules (Gee et al., 1998). In the case of dynein, vesicle binding is thought to be mediated by the interaction of the dynein intermediate chains (ICs) with the 150 kDa Glued subunit of a linker complex, dynactin (Vaughan and Vallee, 1995; Waterman-Storer et al., 1995). Overexpression of the 50 kDa ‘dynamitin’ subunit of the dynactin complex was found to dissociate the complex. Under these conditions dynein was released from its cargo sites (Echeverri et al., 1998), resulting in inhibition of minus end-directed vesicle transport (Burkhardt et al., 1997). In the case of kinesin, cargo binding has been reported to occur through a membrane surface protein, kinectin (Toyoshima et al., 1992; Futterer et al., 1995; Kumar and Schertz, 1995). Cargo binding is thought to occur through the carboxy-terminal end of the kinesin heavy chain, possibly with the participation of the kinesin light chains.
Blocking dynein function, by overexpression of dynamitin, abrogates retention of the class II-containing lysosomes in the MTOC region unless kinesin is inactivated as well. This suggests that the normal intracellular distribution of class II structures, with the bulk of MIIC accumulated in the MTOC area and few transporting vesicles in the periphery, is mediated by the action of two opposing motors. Retaining MIIC in the MTOC area is an active process and requires dynein activity to be superior over kinesin activity on its membranes. Treatment of cells with the protein kinase inhibitor staurosporine induces the dispersion of MIIC, which could imply that phosphorylation events alter the delicate equilibrium of the motors acting on the membranes resulting in loss of retention of the vesicles. The change in direction of MIIC transport and its stop-and-go nature may reflect frequent exchange of vesicle-bound motors during the transport process.
Materials and Methods
Cell culture
Mel JuSo cells were maintained at 37°C in 5% CO2in standard culture conditions (Iscoves
medium, 2 mM glutamine, 100 i.u./ml penicillin, 100 µg/ml streptomycin). Medium for transfectants was supplemented with 500 µg/ml G418 (all obtained from Gibco). Cells were seeded on 24 mm glass coverslips at least 2 days before the experiment in medium lacking G418 and were transferred to a 37°C incubator without CO2 at least 1 hour before the
experiment. During the experiments cells were cultured in Iscoves medium in a coverslip holder (Ince et al., 1985), which was kept at 37°C. To disrupt microtubules, cells were treated with nocodazole (10 µg/ml, Biomol, USA) for 7 minutes. Cells were cultured in the presence of staurosporine (1 µM, Biomol, USA) for 10-20 minutes at 37°C and analysed either directly by confocal laser scanning microscope (CLSM) or after fixation in ice-cold methanol for 2 minutes prior to immunodetection of markers proteins.
Antibodies
The following antibodies were used: rabbit polyclonal anti-Ii C terminus (ICC5, provided by Dr Morton; Morton et al., 1995), mouse monoclonal anti-Ii (VicY1, provided by Dr Knapp, Vienna; Quaranta et al., 1984), rabbit polyclonal anti-CD63 (Vennegoor et al., 1985), rabbit polyclonal HLA-DMA (FS2; Sanderson et al., 1994), rabbit polyclonal anti-mannose 6-phosphate receptor (M6PR, provided by Dr B. Hoflack, Lille; Hoflack and Kornfeld, 1985), mouse monoclonal anti-TfR (B3/25, Boehringer-Mannheim), rabbit polyclonal anti-TGN (TGN46, a kind gift of Dr Ponnamballan; Prescott et al., 1997) and mouse monoclonal anti-dynamitin (H50.1; Echeverri et al., 1998). Antibodies were diluted in phosphate-buffered saline containing 0.5% BSA. Texas Red-conjugated goat anti-rabbit antibodies were purchased from Molecular Probes (Molecular Probes, Leiden).
Cloning
pCMVȕ vector using the BamHI/NotI sites. The NLS-GFP construct was generated by the addition of the nuclear localisation sequence of Xenopus nucleoplasmin (NLS, KRPAATKKAGQAKKKK) upstream of the EGFP cDNA (Clontech) and a downstream SalI site, using PCR. The PCR product was SalI-digested and cloned into the EcoRV/XhoI sites of pcDNA3. Dynamitin cDNA was obtained in the expression vector pCMVȕ (Echeverri et al., 1998).
Image acquisition and analyses
Confocal images were collected by a Biorad MRC600 (Biorad, Hercules, CA) equipped with an Argon/Krypton laser and a heated culture chamber (Ince et al., 1985). Single channel green fluorescence was detected at Ȝ >515 nm after excitation with Ȝ 488 nm. For dual analyses green fluorescence was detected at Ȝ 520-560 nm and red fluorochromes were detected at Ȝ >585 nm after excitation with Ȝ 488 nm and 568 nm, respectively. Timed lapse recordings were analysed as described (Wubbolts et al., 1996). Briefly, 36 images taken every 5 seconds were superimposed and the first image was subtracted. The resulting image was colour coded and merged with the first image. Velocities were calculated using NIH image by collecting pixel positions of peripheral class II-containing structures in the image series (with minor alterations of the kinematics and Biorad import macros from the NIH image homepage: http://rsb.info.nih.gov/nih-image). In Fig. 1A pixel positions were plotted as x-y diagrams in Microsoft Excel and positioned over the inverted first image of the series. Histograms of Fig. 2 were generated by calculating distances between the position of MIIC in successive images. Pixel values were converted into distances using the magnification factor of the microscope and the CLSM settings (Biorad) rendering 19 velocity values per trail of 20 images. Frequencies of individual velocities in the data set were plotted as percentage of the maximal occurrence. Note that this analysis intrinsically results in underestimated speed values, since movement in the z-direction is not taken into account.
M icroinjection
Cells were microinjected on a heated xy-stage of a Zeiss-Anxiovert 135M microscope equipped with an Eppendorf manipulator 5171/transjector 5246 system. Routinely, 50-100 cells were injected intranuclearly with a mix of the cDNA encoding NLS-GFP (1 ng/µl) and the cDNAs indicated (0.1-0.2 µg/µl) in a solution of 120 mM potassium glutamate, 40 mM KCl, 1 mM MgCl2, 1 mM EGTA, 200 µM CaCl2, 10 mM Hepes and 40 mM mannitol, pH
7.2. After injection, cells were cultured at 37°C, and after 3-5 hours either living cells were analysed by CLSM or cells were fixed with 3.7% formaldehyde, permeabilized (by 0.1% Triton-X100 in PBS) and immuno stained before CLSM analysis.
Results
MHC class II-containing vesicles move bidirectionally in a ‘stop-and-go’ fashion
Intracellular accumulation of MIIC in the perinuclear region suggests that a rate-limiting process in the transport of these structures is taking place. We observed living cells at a high magnification to study the details of transport of individual MIIC by collecting images of cells every 3 seconds. Fig. 1A shows cells (only half cells are depicted and the position of the nucleus is indicated by ‘n’) in which representative movement patterns of two individual vesicles are shown. Every point within the trail represents the position of the vesicle in successive images (Fig. 1A). Vesicles move in a ‘stop-and-go’ fashion (arrows indicate the vesicle position in the first image and asterisks are placed where the spacing between points are small indicating ‘stops’). Strikingly, peripheral vesicles frequently return to the MTOC area (grey trail). Furthermore, vesicles are seen that move away from the perinuclear sphere, stop, and return again (and vice versa, as exemplified by the white trail). While approximately 20% of the fluorescent structures are located in peripheral regions only a portion show rapid movement over long distances (1-5% of total, i.e. 3-5 vesicles per minute). At MTOC and peripheral locations ‘steady state’ pools of MIIC seem to be present while the rapid moving pool shuttles between these two locations.
Bidirectional movement occurs in the MTOC area as well (Fig. 1B). Minor oscillations continuously occur, with occasionally an MIIC escaping at high speed (indicated by the arrow at t=3 seconds), and rapidly returning to approximately the same position at t=9 seconds. Images are generated using a frame-averaging method and the ‘trail’ formed at t=3 seconds represents rapid movement of the vesicle during image capture (a similar effect is seen to a lesser extent at t=6 seconds).
Thus, class II-containing structures move in a ‘stop-and-go’ fashion in two directions, away from the MTOC region and returning to this area. Apparently, both the direction and speed of class II-containing vesicles during transport can be modified along the transport route.
MIIC move along microtubules
Since most vesicular transport is supported by microtubules, we analysed movement of MIIC before and after incubating the class II/GFP-expressing cells with the microtubule-depolymerizing agent nocodazole. Images were collected every 3 seconds for a period of 1 minute and the speed of individual movements was calculated (Fig. 2). In untreated cells MIIC move with velocities up to 0.8 µ m/s (upper panels), while nocodazole treatment abrogates the rapid movement (lower panels). This is also represented by the nocodazole-treated cells leaving only small and undirected changes in the class II vesicle positions (data not shown).
Fig. 1. MHC class II-containing MIIC move bidirectionally.
Kinesin mediates plus end-directed transport of class II-containing structures
To test whether the ubiquitously expressed kinesin (Navone et al., 1992) is the plus end-directed motor involved in transport of MIIC, kinesin mutants were generated and overexpressed in the class II-GFP transfectants by nuclear microinjection of cDNAs. The motor domain of kinesin was deleted (aa 1-334, ǻM-KHC; Kozielski et al., 1997; Vale and Fletterick, 1997; Woehlke et al., 1997) to produce a motor that should be unable to drive vesicle movement, while it would still bind cargo. Upon overexpression, ǻM-KHC should interfere with endogenous kinesin function in a dominant-negative fashion.
cDNA encoding the marker protein GFP attached to the nuclear localisation signal (NLS) of Xenopus nucleoplasmin (NLS-GFP) was used to locate injected cells and verify their viability. NLS-GFP was efficiently targetted to the nucleus and therefore did not interfere with the visualisation of class II/GFP-containing structures. Microinjecting low amounts (1 ng/µl) of NLS-GFP cDNA yielded cells with a detectable green nucleus as early as 1.5 hours after injection. Cells coinjected with the cDNA of NLS-GFP and a cDNA encoding the kinesin mutant (present in 200-fold excess) coexpressed both proteins as assessed by immunostaining (data not shown). The migration of class II vesicles was subsequently recorded in living cells after microinjection (Fig. 3). Movement was analysed by the projection procedure described above: the first image of the series shown (left panels) and the movement over 3 minutes is shown as trails (right panel). Microinjection of empty vector (V) and overexpression of wild-type kinesin (KHC) resulted in no obvious effects on distribution and mobility of MIIC, since similar trails are formed in the injected and surrounding cells.
The introduction of kinesin with a deletion of the motor head (ǻM-KHC) strongly inhibited movement of MIIC, leaving only minor vesicle oscillations in the injected cells similar to those observed in cells with disrupted microtubules (Fig. 2). Injecting the motorhead-deleted construct did not affect fluid phase uptake and internalisation followed for up to 30 minutes (data not shown), a period in which late endosomes are accessed (Tulp et al., 1996). These data suggest that kinesin is the motor that drives plus end-directed movement of MIIC.
Fig. 3. Kinesin is the plus end motor that drives movement of MIIC. MHC class II/GFP transfectants were injected with both NLS-GFP (1 ng/µl) and kinesin mutant cDNAs (0.6 µg/µl) 5 hours prior to analyses. Injected cells are identified by the fluorescent appearance of the nucleus. Early lysosomes are labeled by the class II/GFP chimeric molecules. Movement analyses were performed by collecting images every 5 seconds for 3 minutes. Panels on the left depict the first image of the series (for MHC class II and NLS-GFP). Right panels show vesicle movement shown as trails. Microinjecting empty vector (V) and overexpression of wild-type kinesin (KHC) did not affect movement. In contrast, overexpression of the kinesin lacking the motor head domain (ǻM-KHC) inhibited movement of MIIC in the living cells, leaving only small trails in the injected cells. The boundaries of the cells are indicated by the dotted lines. Bar, 10 µm.
Overexpression of dynamitin resulted in a major redistribution of MIIC. In injected cells, labelled vesicles no longer accumulated in the MTOC region but are relocated to the tips of the cells just beneath the plasma membrane. Apparently, intracellular retention of MIIC is critically dependent on dynein activity. MIIC remained in the tips of cells when cells were cultured for longer periods after injection. This indicates that at least one other rate-limiting process should be taking place in the periphery before plasma membrane embedding of class II occurs.
Fig. 4. Dynamitin overexpression causes redistribution MIIC from the MTOC region to the tips of cells. Transfected cells were microinjected with cDNAs of dynamitin (0.2 µg/µl) and NLS-GFP (1 ng/µl; B, top: 5 ng/µl) and living cells were analyzed by CLSM 3 hours after microinjection. Injected cells are marked by the appearance of fluorescent nuclei (left panels) or by ‘i’. The position of the plasma membrane is indicated by the dotted lines. (A) Control cells show accumulation of class II in perinuclear vesicles (living cells, class II/GFP). Dynamitin overexpression causes redistribution of virtually all class II vesicles to the tip of the cells and loss of perinuclear vesicle accumulation. (A) Dynamitin protein was immunodetected (A, p50) as well as marker proteins for trans-Golgi network (A, TGN46), late endosomes (A, M6PR). Late endosomes are relocated whereas the TGN is not. Dynamitin gradually relocates MIIC to the tips of cells. Mel JuSo cells were injected with cDNA of dynamitin mixed with NLS-GFP and fixed after 1, 2 and 3.5 hours of culture at 37°C. The early MIIC marker, Ii was immunodetected (B) and injected cells are shown (indicated by the fluorescent nuclei). Note the massive tip accumulation at 3.5 hours, which is only partial at 2 hours after microinjection. Antibodies used are TGN46, M6PR and H50.1. Bars 10 µm.
Relocation of vesicles as a result of dynamitin overexpression not only affected MIIC, but also late endosomes (positive for M6PR) in the injected cells, which also appeared relocated (Fig. 4A). The trans-Golgi network (TGN) disperses after overnight expression of dynamitin (Burkhardt et al., 1997), but was not fragmented 3 hours after injection of dynamitin cDNA (Fig. 4B, TGN46).
complete at 3.5 hours. These results showed that abrogation of minus end-directed dynein activity mediates rapid relocation of vesicles from the MTOC towards the plus end of microtubules and that dynein is involved in retaining MIIC in this area.
Dynamitin-induced relocalization of class II-containing structures is mediated by kinesin
Retention and transport of MIIC thus appears to be dependent on the activity of two motor proteins, dynein and kinesin, acting in opposite directions. Maintaining dynamitin-induced dispersal was shown to require microtubules (Burkhardt et al., 1997). To directly show that kinesin drives plus end movement of MIIC upon dynein inactivation, the relocation of vesicles caused by dynamitin overexpression was studied in the presence or absence of overexpressed wild-type (KHC) or motor head-deleted (ǻM-KHC) kinesin (Fig. 5). Kinesin cDNA had to be microinjected in excess since it required higher concentrations than dynamitin to exert its effect.
Therefore, the minimal concentration of dynamitin cDNA that still induced relocation of MIIC in 5 hours was determined (Fig. 5, dynamitin). This concentration of dynamitin cDNA was coinjected with excess cDNA encoding the kinesin mutant and cells were analysed after 5 hours culture at 37°C. Injection of the dynamitin cDNA induced relocation, but obvious tip accumulation was not yet observed. Coexpression of the wild-type kinesin protein did not alter the distribution (Fig. 5, dynamitin+KHC). However, coexpression of kinesin lacking the motor domain resulted in a clear abrogation of the dynamitin-induced relocation of MIIC while dynamitin was expressed at equal levels (Fig. 5, dynamitin+ǻM-KHC). Light fluorescent reticulated staining seen in the injected cell may reflect class II trapped in the ER, since ER-to-Golgi transport is dynein-dependent and intra-Golgi transport is kinesin-dependent as well (Lippincott-Schwartz et al., 1995). MIIC were still located in the perinuclear region, as was the case for control cells and cells expressing the kinesin proteins only. This indicated that the redistribution of MIIC upon dynein inhibition was mediated by kinesin. Apparently, dynein activity determines retention of the class II-containing structures in the MTOC area. Upon inactivation of dynein it is the kinesin motor that transports MIIC to the periphery.
Staurosporine causes redistribution of MIIC
Apparently, both kinesin and dynein are able to affect MIIC retention. They could be the ultimate targets for regulatory processes in class II transport, either by altering motor activity or by modulating vesicle binding. To study the regulation of MIIC transport, we tested whether pharmacological agents could affect the distribution of class II vesicles. Agents that increase intracellular Ca2+levels (thapsigargin, ionomycin) do not show effects on the class II distribution. AlF4, a compound that activates GTP binding proteins, also had no detectable effect (data not shown). Not surprisingly, the MIIC transport requires ATP, since NaN3/2-deoxyglucose treatment of the cells blocked movement of MIIC, in line with transport along microtubules propelled by motor proteins (data not shown). Treatment of cells with the protein kinase inhibitor staurosporine (1 µM) rapidly increased MIIC appearance in the periphery (Fig. 6B), while tyrosine kinase inhibitors (genistein, herbimycin A and tyrphostins 25/47) did not induce these effects. As suggested by the relatively high concentration necessary to mediate redistribution, the effect of staurosporine most probably was not due to an inhibition of (classical) PKCs (IC50 in the nM range) since long-term TPA pre-treatment did not mimic the staurosporine effect.
Fig. 6. Staurosporine induces microtubule-dependent redistribution of MIIC. Living class II/GFP transfectants were observed by CLSM before (A) and after (B) treatment with staurosporine (1 µM) for 20 minutes at 37°C. Staurosporine treatment causes redistribution of the class II vesicles. Pre-treatment with nocodazole (C, 5 µg/ml, 30 minutes) blocked staurosporine-mediated redistribution (D). Note that staurosporine treatment decreases perinuclear accumulation of class II vesicles in the cells and increases the number of peripheral vesicles. Bars 10 µm.
To study whether only MIIC were affected by the staurosporine treatment, several markers for intracellular compartments were immunodetected before (Fig. 7C) and after staurosporine treatment (Fig. 7st). The location of trans-Golgi network (TGN), early endosomal compartments (labelled by the transferrin receptor, TfR) and late endosomes (marked by mannose 6-phosphate receptor, M6PR) are not detectably affected by staurosporine. In contrast, the lysosomal markers (CD63 and lamp-1 (not shown)) and the MIIC markers invariant chain (Fig. 7ICC) and DM (Fig. 7DMA) relocated after staurosporine treatment, resulting in peripheral accumulation of MIIC vesicles. Since staurosporine affects lysosomal-like compartments while the TGN, early and late endosomes are not affected, a protein kinase-dependent event is probably required for the regulation of vesicular transport of MIIC and lysosomes.
Discussion
Fig. 7. Effect of staurosporine on intracellular compartments. Cells were cultured in the absence (C) or presence of staurosporine (st, 1 µM for 20 minutes), fixed, and markers for the trans-Golgi compartment (TGN46), early endosomes (TfR), late endosomes (M6PR), class II vesicles (DMA and ICC) and lysosomes (CD63) were immunolocalized. The positions of trans-Golgi compartments (TGN) and earlier endosomal structures (TfR, M6PR) are unaffected but MIIC are redistributed. Bar 10 µm.
Transport of class II-containing structures has been studied by following the class II/GFP-labelled vesicles in real time. Here, we have analyzed the transport of class II structures in living cells in detail. MIIC are accumulated in a region around the MTOC and the Golgi complex, located proximal to the nucleus. Occasionally, an MIIC is released from this area and transported via microtubules away from the MTOC. However, the release is not definite and vesicles often return to their original location by oppositely directed movement. Moving MIIC in the periphery do not lose fluorescence or reduce in size, indicating that the integrity of MIIC is maintained during transport. The bidirectional nature of MIIC transport suggests that regulatory events for release from or return to the MTOC region can be made all along the vesicular transport route.
(reviewed in Goodson et al., 1997). Many plus end-directed, microtubule-dependent motors have been identified with different cargo specificity and tissue expression (Hirokawa, 1998). The ubiquitously expressed kinesin has been shown to function in a wide variety of organelle transport processes (Goodson et al., 1997) including lysosomes (Hollenbeck and Swanson, 1990; Swanson et al., 1992; Perou and Kaplan, 1993; Nakata and Hirokawa, 1995; Rodriguez et al., 1996).
We overexpressed a dominant-negative mutant of kinesin to study its involvement in class II transport. Nuclear microinjection of cDNA results in rapid expression (here 3-6 hours after injection) of the encoded protein, allowing a study of effects of potentially lethal proteins. We conclude that kinesin is involved in MIIC transport to the plus end of microtubules since overexpression of kinesin lacking the motor domain inhibited movement of MIIC whereas no effect was observed upon overexpression of wild-type kinesin. Velocities observed for the MIIC transport (with maximal measured speeds of 0.8 µm/second) are comparable with speeds obtained for kinesin-mediated motility and correspond to approximately 100 steps of the motor per second (Schnitzer and Block, 1997).
MIIC move towards the minus end of the microtubular track as well, an activity that can be driven by the motor protein dynein (Paschal et al., 1987; Schroer et al., 1989; Holzbaur and Vallee, 1994). Dynein function has been implicated in endosomal transport (Aniento et al., 1993; Oda et al., 1995) and in one study, dynein was shown to be associated, not necessarily functionally, with lysosomes (Lin et al., 1996). Overexpression of dynamitin, a subunit of the multiprotein complex dynactin that links dynein to its cargo, dissociates this complex and consequently inhibits dynein-mediated movement (Echeverri et al., 1998) and results in the redistribution of various subcellular structures including lysosomes (Burkhardt et al., 1997).
The retrograde transport of MIIC requires the action of dynein, as inhibition of its activity in class II/GFP transfectants by overexpression of dynamitin abrogated fast MIIC movement (data not shown) and virtually all MTOC-accumulated vesicles were relocated to the tips of the cells. This shows that dynein inhibition affects the plus end transport as well, a feature that is described for microtubule-based transport mechanisms (Hamm-Alvarez et al., 1993). This is supported by the kinetics of the relocation process, which is complete only 3.5 hours after microinjection of dynamitin cDNA. However, kinesin activity is required for the relocation process (Fig. 5). The data imply that dynamitin overexpression interferes with the transport process itself by, for example, reducing the amount of steps an MIIC-bound kinesin molecule is able to make.
Apparently, the retention of MIIC in a perinuclear region requires functional dynein while the opposing action of kinesin mediates the transport of the released structures and maintains them in the tip. This is supported by the simultaneous expression of dynamitin and mutant kinesin. Expression of mutant kinesin was able to block the relocation of MIIC to the tips of the cells when dynamitin is expressed as well.
In conclusion, the fate of MIIC in the MTOC region is determined by both kinesin and dynein action and the control of MIIC transport to the periphery may ultimately result in either activation of kinesin and/or inactivation of dynein, by for example dissociation of the dynactin complex. Alternatively, the affinity for the motor proteins to bind their receptors (Toyoshima et al., 1992; Futterer et al., 1995; Yu et al., 1995; Leung et al., 1996) on the MIIC is altered, resulting in dislocation of dynein and increased binding of kinesin. The accumulation of MIIC in the tips of dynamitin-overexpressing cells just beneath the plasma membrane suggest that at these locations another rate-limiting event occurs in the transport of MIIC and cell surface deposition of class II molecules.
How activities of motor proteins or their binding affinities to cargo can be regulated is unclear. Multiple mechanisms have been proposed, many of which involve phosphorylation events on serine/threonine residues of either the kinesin and associated proteins (Hollenbeck, 1993; Lee and Hollenbeck, 1995; Lindesmith et al., 1997), or dynein/dynactin (Lin et al., 1994; Farshori and Holzbaur, 1997), or both (Hamm-Alvarez et al., 1993). Microtubule-associated proteins (MAPs) have been suggested to compete for motor binding to microtubules in a phosphorylation-dependent manner as well (Lopez and Scheetz, 1993; Hagiwara et al., 1994; Sato-Harada et al., 1996). We have tested a variety of pharmacological compounds to interfere with the retention process in MIIC transport by analyzing morphological changes in the distribution of the vesicles. The protein kinase inhibitor staurosporine rapidly induces the depletion of perinuclear vesicles, while simultaneously many peripheral vesicles appear. Staurosporine appears to be more selective for MIIC and lysosomes than dynamitin overexpression, which affects the distribution of many other compartments as well (Burkhardt et al., 1997). Interestingly, other tyrosine kinase inhibitors (herbimycin A, genistein, tyrphostins 47 and 26), myosin light chain kinase inhibitors (ML-7 and KT5926) and long term pre-treatment with TPA, which inhibits classical PKCs, did not mimic the effect seen in staurosporine-treated cells (data not shown). A cytosolic factor important for in vitro minus-end movement of phagosomes was found to be sensitive to staurosporine inhibition as well (yet sensitive in the nM range). Interestingly, this factor could be immunodepleted from cytosol with antibodies recognising Arp1, a subunit of the dynactin complex (Blocker et al., 1997).
Recently, the first indications for regulation of class II transport have been obtained, in particular dendritic cells (DC) alter the distribution of class II molecules upon maturation (Sallusto and Landzavecchia, 1994; Nijman et al., 1995; Cella et al., 1997; Pierre et al., 1997) and IL-10 rapidly inhibits transport of both internalised and the newly synthesised molecules from class II compartments to the plasma membrane (Koppelman et al., 1997). Recent studies suggest a role for the invariant chain in the regulation of intracellular positioning of class II (Anderson and Roche, 1998; Pierre and Mellman, 1998). Effective breakdown of this class II specific chaperone (Cresswell, 1996) by cathepsin S (Reise et al., 1996; Deussing et al., 1998) confers location of class II to earlier endosomal locations than lysosomes in maturing dendritic cells (Pierre and Mellman, 1998). Moreover, Ii is phosphorylated in a staurosporine-sensitive manner which also delayed endosomal targeting of class II molecules (Spiro and Quaranta, 1989; Anderson and Roche, 1998). Although the exact molecular basis for these effects remains unclear, they ultimately should interfere with the motors involved in transport of class II vesicles to the plasma membrane.
opposite-directed motor proteins in class II transport may be a general mechanism for regulating intracellular organisation and transport of various compartments, including the maturation of phagosomes in macrophages (Blocker et al., 1997). Remarkably, retention of MIIC in the MTOC region is apparently an active process and requires the continuous activity of dynein motors. The molecular basis for the control of the respective motor activities will further add to our understanding of the timing and regulation of class II transport upon dendritic cell maturation, by IL-10 signalling and potentially many other factors that modulate vesicular transport in cells.
Acknowlegdments
We thank Lauran Oomen and Ilja van de Pavert for excellent support on CLSM and image data analyses, and Dr Ron Vale for the kinesin reagents. We also thank Marieke van Ham, Chris Vos and Kevin Vaughan for critically reading the manuscript. This research has been supported by EEC grant PL960505 (R.W), TMR grant FMRXCT960069 (M.F.-B.) and Pioneer grant from the Dutch Society of Scientific Research NWO (J.J.N., S.D. and E.R.)
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